Name: retrograde neuroanatomical tracing of phrenic motor neurons in mice
Uploaded: 2018-02-22T14:00:00
Description: Watch this Scientific Journal Video about Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice at JoVE.com

We are grateful to Robert Graffin and Pauline Duhant for their technical support.

The experimental protocol was conducted in compliance with the European Communities Council Directives for Animal Experiment (2010/63/EU, 86/609/EEC and 87-848/EEC) and was approved by the Animal Ethics Committee of University of Namur (ethic project n&#176;17-284). Figure 1 depicts the two respective approaches: transdiaphragmatic or transthoracic injections. Use male C57bl/6J mice (n=18), aged from 3 to 4 months in the study.
1. Preparation of CTB Solution
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The protocol described herein can be applied to any strain of adult mice or to any experimental paradigm in which the integrity of the diaphragm-PhMN circuitry should be evaluated. For instance, amyotrophic lateral sclerosis (ALS) and cervical spinal cord injury (cSCI) are conditions associated with PhMN loss, anterograde degeneration of phrenic axons and subsequent respiratory compromise. Animal models of ALS or cSCI mimic histopathological and functional respiratory deficits observed in human diseases. In these models,...

The aim of the study is to present a reliable method to identify phrenic motor neurons (PhMN) on mouse spinal cord sections. Injection of a fluorescent neuroanatomical tracer in the pleural cavity was chosen as the delivery method to reach the phrenic neuromuscular projections onto the diaphragm and use retrograde transport along the phrenic axons to label phrenic cell bodies. Two techniques of intrapleural delivery are described: transdiaphragmatic versus transthoracic.
Phrenic motor neurons are spinal relay cells whose axons converge into phrenic nerves, which ultimately innervate the diaphragm. These are lower motor neurons receiving the inspiratory drive from the bulbar respiratory centers and relaying it to the diaphragm neuro-muscular junctions (NMJ). PhMN are structured into two motor columns, one for each hemicord, running along the mid-cervical spine. In most of mammalian species including humans, the phrenic motor columns spread from levels C3 to C61,2,3. We and others have confirmed that PhMN concentrated in C3-C5 levels in rat and mouse spinal cord4,5,6,7,8. The topographical distribution of phrenic cells is not at random; motor neurons innervating the sternal part of the diaphragm are distributed more densely in the cranial part of the phrenic motor pool (C3), whereas motor neurons innervating the crural part are more caudal (C5)9. Furthermore, PhMN are clustered variously in the ventral horn gray matter. At C3 level, the clusters of phrenic cells lie laterally, then they shift in a ventrolateral direction and are found ventromedially at the most caudal levels10,11.
Given their vital role during inspiration, it is of the utmost importance to accurately identify PhMN in the healthy spinal cord but also follow their fate during pathological conditions, such as degenerative diseases or traumatic injuries of the spinal cord. Since PhMN do not differ morphologically from other cervical motor neurons, identification of PhMN relies on the targeted delivery of neuroanatomical tracers either at the level of primary respiratory centers8, or at the diaphragm NMJ7 or in the phrenic nerve4. The tracer is taken up by the nerve fibers and carried up to the phrenic cell bodies in the cervical spine, where it can be visualized using direct or indirect detection systems. Retrograde or anterograde tracers are commercially available with a broad range of conjugates. Noteworthy, each tracer is endowed with no, low or high abilities for trans synaptic tracing.
In the current study, we chose the beta subunit of the cholera toxin (CTB) functionalized with Alexa Fluor 555 (henceforth referred to as CTB-fluorophore) as a fluorescent label, allowing a direct visualization of PhMN on frozen spinal cord sections. CTB is usually described as a monosynaptic tracer although experimental data tend to show a transneuronal passage12. CTB has the ability to bind the ganglioside GM1 at the plasma membrane of the nerve ending. CTB is internalized via clathrin-dependent or -independent mechanisms and traffics through the trans-Golgi network into the endoplasmic reticulum in a retrograde fashion13,14. The internalization and retrograde transport seem to be dependent on the actin cytoskeleton15,16 as well as on the microtubule network17.
To demonstrate the usefulness of CTB as a retrograde neuroanatomical tracer labeling diaphragm-PhMN circuitry, CTB-fluorophore was delivered intrapleurally. CTB was administered using two techniques: the first one included a laparotomy and multiple transdiaphragmatic injections; the second one, less invasive, used a unique transthoracic injection. Four days later, fluorescently-labeled PhMNs were quantified in the cervical spinal cord from both from healthy and from spinally-injured (C4) animals.

<table>
	<tbody>
		<tr>
			<td>Glass-bead sterilizer Steri 250</td>
			<td>Keller</td>
			<td>31-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Small scissors</td>
			<td>F.S.T.</td>
			<td>14058-00</td>
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			<td>Soft tweezers</td>
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			<td>11042-08</td>
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			<td>Scalpel blades</td>
			<td>Swann Morton</td>
			<td>No.11 or 15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera toxin subunit beta conjugated to Alexa Fluor 555</td>
			<td>Life Technologies</td>
			<td>C22843</td>
			<td>Bring at room temperature before use&#160;</td>
		</tr>
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			<td>10ul Hamilton syringue, removable needle</td>
			<td>Sigma-Aldrich</td>
			<td>701RN</td>
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			<td>9mm autoclips</td>
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retrograde neuroanatomical tracing of phrenic motor neurons in mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Male C57bl/6J mice (n=18), aged from 3 to 4 months were included in the study. At day 0 of the experiment, 8 mice underwent a unilateral C4 contusion, right-sided, according to published protocol7,18. As sham procedure, 10 mice underwent a laminectomy on top of C4 without contusion. At day 3, mice were prepared for the intrapleural injections of CTB-fluorophore according to the two different procedures described above. At day 7, a...

Watch this Scientific Journal Video about Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice at JoVE.com

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

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